The present invention relates generally to the field of recombinant proteins, compositions, vectors, kits, and methods for immunizing against coronaviruses. In particular, the invention relates to recombinant infectious bronchitis virus (IBV) spike ectodomain proteins, compositions, vectors, kits, and methods for immunizing avian species against infection by IBV.
In the poultry industry avian infectious bronchitis virus (IBV) continues to be the most common contributor to respiratory disease in chicken populations despite worldwide extensive vaccination with a multiplicity of type-specific vaccines. IBV replicates primarily in the upper respiratory tract causing respiratory disease in large chicken populations. IBV's surface spike (S) glycoprotein is post-translationally cleaved into an S1 subunit (˜550 amino acids) and an S2 subunit (˜600 amino acids) (Lai and Holmes, 2001). Like other coronaviruses, the S1 subunit of the S glycoprotein is responsible for viral attachment to host cells and is important for host protective immune responses as it induces virus neutralizing-antibodies (Cavanagh, 1981, 1983, 1984; Cavanagh and Davis, 1986; Koch et al., 1990; Koch and Kant, 1990; Mockett et al., 1984). Because of the relevance of S1 for the first step of replication (i.e., attachment to cells) and immunological escape, the extensive variation exhibited by The S1 glycoprotein among IBV coronaviruses (Kusters et al., 1987; Kusters et al., 1989b) is likely the most relevant phenotypic characteristic for this virus's “adaptation” and evolutionary success (Toro et al., 2012b). Genetic diversity among coronaviruses is achieved by high mutation frequency and recombination events (Enjuanes et al., 2000a; Enjuanes et al., 2000b; Lai and Cavanagh, 1997; Stadler et al., 2003). Selection acting on diverse populations results in rapid evolution of the virus and the emergence of antigenically different strains (Toro et al., 2012b). More than 30 different IBV types have been identified during the last 5 decades in the U.S. alone. According to a 2012 review, over 30 different genotypes of IBV are currently affecting chicken populations worldwide (Jackwood, 2012). These have recently been grouped into 6 genotypes divided into 32 lineages (Valastro, V., E. C. Holmes, P. Britton, A. Fusaro, M. W. Jackwood, G. Cattoli, and I. Monne. S1 gene-based phylogeny of infectious bronchitis virus: An attempt to harmonize virus classification. Infect Genet Evol 39:349-364. 2016). Multiple surveillance studies performed in the U.S. have demonstrated that serotypes/genotypes Arkansas (Ark), Massachusetts (Mass), Connecticut (Conn), DE072, and Georgia variants GAV and GA98 are the most prevalent (Jackwood et al., 2005; Nix et al., 2000; Toro et al., 2006). The scientific literature states that the amino acid sequences of the S1 subunit of different IBVs can have as little as 50% amino acid sequence identity (Valastro et al., 2016). However IBV S1 amino acid sequences with even less (˜45%) amino acid sequence identity can be found in GenBank. The amino acid sequences of IBV S2 subunits are more conserved than those of S1 subunits; nevertheless considerable variation is found. One report indicated that IBV S2 subunits exhibited as little as 74% amino acid identity (Toro et al., 2014). A more recent analysis of IBV S2 ectodomain amino acid sequences in GenBank showed some with as little as 70% amino acid sequence identity.
Because IBV exists as multiple different serotypes that do not provide for cross-protection after host exposure or vaccination, a multiplicity of serotype-specific IBV vaccines have been developed worldwide. For example, vaccination programs in the U.S. currently comprise mono- or polyvalent vaccines including Mass, Conn, GA98, DE072, and Ark serotypes. In Europe, IBV vaccines commonly include strains belonging to serotypes Mass, UK4/91, D274, and D-1466. However, IBV's high ability to evolve allows it to consistently circulate in commercial poultry and cause outbreaks of disease in spite of extensive vaccination. In addition, accumulating evidence indicates that attenuated IBV vaccines may also be contributing to the emergence and circulation of vaccine-like viruses in host populations (Toro et al., 2012b; Toro et al., 2012c). Indeed, viral sub-populations differing from the predominant live vaccine population have been shown to emerge during a single passage of attenuated IBV vaccine in chickens (McKinley et al., 2008; van Santen and Toro, 2008).
In an effort to understand the mechanisms underlying the emergence of vaccine-like viruses, S1 gene sequences of virus populations of all four IBV Ark-serotype attenuated vaccines commercially available in the U.S. were analyzed before and after replication in chickens (Gallardo et al., 2010; van Santen and Toro, 2008). The results from these analyses demonstrated different degrees of genetic heterogeneity among Ark-derived vaccines prior to inoculation into chickens, ranging from no apparent heterogeneity to heterogeneity in 20 positions in the S gene. In all except one position, nucleotide differences resulted in different amino acids encoded and therefore in phenotypic differences among subpopulations present in the vaccines. Significantly, it has been observed that specific minor subpopulations present in each of the vaccines were rapidly “selected” during a single passage in chickens. Indeed, by 3-days post-ocular vaccination, viral populations with S gene sequences distinct from the vaccine major consensus sequence at 5 to 11 codons were found to predominate in chickens (Gallardo et al., 2010; McKinley et al., 2008; van Santen and Toro, 2008). Thus, the use of attenuated coronavirus vaccines may be contributing to the problem of antigenic variation, and the development of a novel vaccine technology to increase the resistance of chicken populations to IBV and reduce economic losses is essential for the poultry industry.
The currently most effective and most widely used IBV vaccination program for broilers utilizes live-attenuated vaccines of various serotypes generated by extensive serial passage in embryonated chicken eggs, applied by eyedrop or spray in the hatchery and often a second time by spray or drinking water in chicken houses at 14-18 days of age. Disadvantages of this type of vaccine include the long time required to produce attenuated vaccine strains of emerging serotypes and the risk of increase in virulence as the vaccine strain circulates among inadequately vaccinated chickens in a flock.
The vast majority of efforts to generate vectored vaccines or recombinant subunit vaccines against IBV to overcome the disadvantages of live-attenuated vaccines have utilized sequences encoding the amino terminal portion (S1) of the spike protein. However, here, the inventors have shown that immunization with a larger portion of the spike protein, including the entire ectodomain, provides more effective protection against IBV infection than the S1 domain alone. Importantly, the recombinant proteins disclosed herein may be derived and prepared from any IBV strain in order to generate a more effective antigen for inducing a protective immune response.